Abstract : Plasmodium vivax remains an important cause of morbidity outside Africa, and no effective vaccine is available against this parasite. The P. vivax Duffy binding protein (PvDBP) is essential during merozoite invasion into erythrocytes, and it is a target for protective immunity against malaria. This investigation was designed to evaluate naturally acquired antibodies to two variant forms of PvDBP-II antigen (DBP-I and -VI) in malaria individuals (N = 85; median = 22 years) who were living in hypoendemic areas in Iran. The two PvDBP-II variants were expressed in Escherichia coli, and immunoglobulin G (IgG) isotype composition and avidity of naturally acquired antibodies to these antigens were measured using enzyme-linked immunosorbent assay (ELISA). Results showed that almost 32% of the studied individuals had positive antibody responses to the two PvDBP-II variants, and the prevalence of responders did not differ significantly (P > 0.05; χ(2) test). The IgG-positive samples exhibited 37.03% and 40.8% high-avidity antibodies for PvDBP-I and PvDBP-VI variants, respectively. Furthermore, high-avidity IgG1 antibody was found in 39.1% of positive sera for each examined variant antigen. The avidity of antibodies for both PvDBP variant antigens and the prevalence of responders with high- and intermediate-avidity IgG, IgG1, and IgG3 antibodies were similar in patients (P > 0.05; χ(2) test). Moreover, the prevalence of IgG antibody responses to the two variants significantly increased with exposure and host age. To sum up, the results provided additional data in our understanding of blood-stage immunity to PvDBP, supporting the rational development of an effective blood-stage vaccine based on this antigen.